JP2006516743A - Microchip-based system for HIV diagnostic related applications - Google Patents

Abstract

The present invention relates to a microchip-based assay for measuring HIV-related analytes (eg, CD4 lymphocytes, HIV-RNA, and liver enzymes) in samples taken from subjects infected with HIV virus. The method of the present invention is ideal for use during HIV disease monitoring in resource-poor environments.

Description

Related applications

In this application, U.S. Patent Application No. 60 / 445,143, filed February 5, 2003, U.S. Patent Application No. 60 / 447,070, filed Feb. 13, 2003, and Jul. 24, 2003. It claims the benefit of priority over international application number PCT / US03 / 23131 filed on the day.
Each document cited or referenced in each of the above applications, and any manufacturer's instructions or catalog relating to any product cited or referenced in each of the above applications and all cited references, are hereby incorporated by reference. To do. In addition, manufacturer's instructions or catalogs for all references cited in the text, all references cited or referenced in references cited in the text, and any product cited or referred to in any references incorporated in the text or text. Are hereby incorporated by reference. Literature incorporated by reference or teachings therein can be used in the practice of the invention. Documents incorporated herein by reference are not admitted to be prior art.

  The present invention relates to a microchip-based assay for measuring HIV-related analytes (eg, CD4 lymphocytes, HIV-RNA and liver enzymes) in subjects infected with HIV virus. The method of the present invention is ideal for use during HIV disease monitoring in a scarce environment.

Description of potential government benefits

  The US government may have certain rights in this invention based on grant numbers R21 AI053911-01 and R37 AI28568-13 from the National Institutes of Health.

  Human immunodeficiency virus, or HIV, is the cause of acquired immunodeficiency syndrome (AIDS) and is a very serious global health problem. AIDS was first reported in the early 1980s and is characterized by severe immune dysfunction with various clinical features such as opportunistic infections, tumors, and central nervous degeneration. While significant advances have been made in the treatment of the molecular characteristics of the virus and AIDS-related symptoms, there is much to be done to eradicate HIV-infected individuals. No effective vaccine has been produced so far, and currently marketed drugs have not solved the problem of HIV's frequent and rapid mutation rate.

  HIV is a retrovirus belonging to the family Lentiviridae. There are at least two subtypes of HIV (HIV-1 and HIV-2). HIV-1 is by far the most common subtype in the United States, while HIV-2 is prevalent in West Africa. Infectious HIV particles are composed of two positive-strand RNA molecules, each consisting of a 9.3 kilobase genome. The HIV genome is encapsulated in the core of the viral protein and surrounded by a phospholipid bilayer membrane produced from the host cell. The phospholipid bilayer membrane includes proteins generated from host cells, in addition to proteins of cell membranes encoded by viruses. The structure of the HIV genome is based on nucleotide sequences called gag, pol, env. gag encodes a polyprotein that degrades the protein to produce the core structural protein of the HIV viral particle. Gag is the most abundant protein in the virus particle, accounting for nearly 90 percent of the structural protein of the virus particle. Gag is also the only protein required for the formation of infectious particles, so the association of gag at the host cell plasma membrane is the driving force for virus particle formation. The gag encodes mature protein substrate (“MA”), capsid (“CA”), spacer peptide 1 (“SP1”), nucleocapsid (“NC”), SPI, p6gag. The polyprotein is cleaved by a protease encoded by pol. The pol sequence encodes the reverse transcriptase, endonuclease and viral protease enzymes required for viral genome replication. Pol is expressed by a -1 frame shift during gag translation, synthesizes a gag-pol polyprotein, and when the polyprotein is processed, it reorganizes the structure of the virion after budding. The env sequence encodes the glycoproteins gp120 and gp41 present in the envelope of the viral particle.

  Furthermore, as another gene involved in virus particle formation, vpr, vif, tat, rev, nef, and vpu are included in the HIV genome. tat is a 14 kD protein having transcriptional activity that promotes the expression of the entire HIV gene, and transcription and then translational activity. Rev is a 20 kD protein that stably encodes, processes, and transports a viral messenger RNA molecule that encodes the gag, pol, and env genes and suppresses the expression of tat, nef, and rev itself. nef encodes a prenylated protein whose mechanism of action is unknown. Similarly, the nature of the protein encoded by vpr, vif, vpu is not known, but may play a role in the infectivity of virions.

  HIV-1 and HIV-2 differ in several ways. For example, the HIV-2 subtype expresses an additional gene, vpX, and lacks the vpu gene. Like vpu, vpX is not well characterized. Furthermore, the rev gene of HIV-2 contains a larger insert compared to HIV-1. Finally, since the env genes are different between HIV-1 and HIV-2, the antibody identification method also differs. Therefore, diagnostic tests for HIV need to differ between these two virus types. Furthermore, both HIV-1 and HIV-2 cause considerable mutations in the action of the immune system or antiretroviral drugs. Diagnostic tests also need to be able to identify infinite types of virus subtypes due to such mutations.

  HIV not only initially infects T lymphocytes that express the cell surface antigen CD4, but also infects lymph node macrophages and follicular dendritic cells that function as antigen-presenting cells. The CD4 protein binds with high affinity to the HIV gp120 glycoprotein, which promotes a membrane fusion event and infects the HIV genome from cell to cell. Membrane fusion and internalization of virus particles into cells is promoted via the gp41 protein. Multiple host cell factors also affect the entry of virus particles into the cell. Upon entry, the enzyme in the nucleoprotein complex is activated, the HIV RNA genome is reverse transcribed to produce the corresponding DNA molecule, and integrated into the host cell genome by the integrase encoded by the virus. Integration is also promoted by co-activation of T cells for viral entry, but proviruses may remain transcriptionally inactive for months or years.

  The presence of two long terminal repeats (LTR) at both ends across the structural gene up-regulates transcription of the integrated DNA provirus. These cis-acting sequences are distinguished by host proteins such as nuclear factor κB (NF-κB) and transcription factor SP1. Like other important processes in viral internalization, transcription of viral proteins is facilitated by host proteins and host signal transduction mechanisms. For example, transcription is enhanced by up-regulating small cell factors known as cytokines. Such signaling molecules regulate immune system function by participating in a variety of cellular events including, but not limited to, blood cell differentiation, cell surface antigen expression, apoptosis, antibody antigen recognition, and the like. Cytokines include tumor necrosis factor (TNF), virus suppressors (IFN α, β, and γ), and interleukins. From another perspective, it can be said that the same mechanism that leads to the proliferation and maintenance of CD4-expressing cells also leads to HIV replication.

  Antiretroviral therapy has been plagued by an undeniable fact, namely the rapid and frequent mutation of the HIV genome caused by HIV. While many treatments are directed at one specific viral protein, HIV stores the ability to gradually replicate in several different body compartments while viral replication and processing are strongly suppressed by combination therapy. Yes. In this way, the HIV genome is mutated and eventually can avoid drugs. Current drug and treatment regime protection is ultimately insufficient to control virus replication in the long term. However, there is another research subject that has not yet been explored: the life cycle of viruses. Two main types of drugs are currently available: nucleotide inhibitors that target reverse transcriptase and protease inhibitors that inhibit the proteolytic processing of polyproteins encoded by the viral genome. (Menendez-Arias El. 2002 “Trend in Pharmacology Science” No. 23 (8): p. 381-388 (Menendez-Arias, L. 2002. Trends Pharm. Sci. 23 (8) : 381-388)). As a third type of drug, an invasion inhibitor that inhibits HIV virus particles from fusing with the membranes of CD4-expressing cells was recently added to a prescription approved by the US Food and Drug Administration (FDA).

  Inhibitors of HIV reverse transcriptase ("RT") include nucleoside analog inhibitors ("NRTI" or zidovudine monophosphate), acyclic nucleoside phosphonic acids (i.e. tenofovir), non-nucleoside RT inhibitors ("" NNRTI ", i.e. nevirapine), and pyrophosphate analogs. Nucleoside inhibitors act as competitive inhibitors of HIV-RT substrates, and during the phosphorylation reaction these agents act as chain terminators and prevent elongation of the growing DNA strand. Resistance to this type of inhibitor is caused by mutations in residues close to the nucleotide binding site of HIV-RT, but agents that stop chain elongation, such as zidovudine monophosphate (“AZT”), can be ATP or pyrroline. It also arises from mutations that are to be excluded from DNA primers that have been inhibited through acid-mediated phosphorolysis. Although mutations and amino acids differ between various HIV-RT inhibitors, it is clear that the mechanism of action of all these types of RT inhibitors can be avoided.

  Another drug action target is HIV protease ("PR"). As explained above, proteases are involved in proteolytic processing of proteins encoded by the HIV genome. HIV-PR inhibitors act as competitive inhibitors of proteolytic reactions. Examples include indinavir and saquinavir. Major resistance mutations are generally found in residues contained in the substrate binding pocket. These mutations reduce protease catalysis and ultimately reduce viral replication. However, additional mutations supplement the proteolytic function of this enzyme. Treatment with both PR and RT inhibitors has had limited success with regard to viral replication suppression, but combination therapy with multiple drugs is compared to treatment with just one drug or one drug. Effective for different ranges of mutations. Other targets for the viral life cycle include gp41 protein and integrase as important research themes. One agent that can currently be used as one agent that targets the gp41 / gp120 molecule and prevents fusion is an invasion inhibitor (enfuvirtide).

  While progress has been made in recent years with regard to the introduction of effective antiretroviral therapies in the world's poorest countries, most of these countries have been severely damaged by the spread of HIV. In May 2002, US $ 616 million was provided as a Global AIDS, Tuberculosis and Malaria Countermeasures Fund, and 58 projects were supported in 37 countries, 70% of which covered HIV / AIDS. (Global Fund to Fight AIDS, Tuberculosis, and Malaria, Global Fund Update, June 2002). Other local initiatives include Clinique Bon Saveur in Central Haiti, which can and should be treated as a standard treatment for people living with HIV even in the most remote areas. (Farmer P.E. 2002, 14th International AIDS Conference). Nevertheless, of the approximately 25 million people in developing countries who need antiretroviral treatment, only 50,000 are currently receiving treatment, and only 40,000 are targeted by the 2003 World Fund Project. (Global AIDS, Tuberculosis and Malaria Countermeasure Fund “Global Fund Update” June 2002 (Global Fund to Fight AIDS, Tuberculosis, and Malaria, Global Fund Update, June 2002)).

  An important issue that the World Fund and HIV treatment program advocates in most developing countries have not solved is the lack of inexpensive HIV clinical tests necessary to monitor treatment progress. Because there are financial and structural barriers to conducting HIV clinical testing in a scarce environment, the obstacles faced by drug procurement programs sometimes appear trivial. In the treatment program, the average annual total medical cost per person is $ 45 (Musgrove P. et al., 2002 “Britin of World Health Organization” No. 80; p. 134-146 (Musgrove , P. et al, 2002. Bull. World Health Organ. 80: 134-146)), the cost of HIV clinical testing is about one year per patient in developing countries. $ 140 to $ 1,500 (Floyd K. and C. Gilx, 1997, World Health Organization; Stewartlander B. et al., 2001 "Science" No. 292: p. 2434-6 (Floyd, K. and C. Gilks, 1997. World Health Organization; Schwartlander, B. et al, 2001. Science 292: 2434-6)). In addition, current HIV clinical testing requires sophisticated research equipment, trained technical staff, and stable power and refrigeration, all of which are scarce resources in most countries with severe HIV infection. . It is important to pay equal attention to the question of how to monitor HIV-infected patients worldwide if the treatment program is effectively implemented in a scarce environment.

  Serological analysis to detect HIV antibodies remains the only widely used HIV test as a means of diagnosing HIV infection. More than 24 million HIV serologic tests are conducted in the United States annually (Centers for Disease Control and Prevention, Annual Report 1997-1998. June 2001) ), And a huge number of tests are being carried out around the world. In the United States and developing countries, after screening by enzyme-linked immunosorbent assay (ELISA), if the ELISA is positive, confirmation by Western blotting is performed. Rapid ELISA has been developed because cheaper and faster serum tests are required in resource-poor environments. An ELISA can be performed for a total cost of $ 3 to $ 5 per patient.

  Serum tests for diagnosing HIV have been standardized in the mid 1980s, but monitoring clinical progress of HIV infection has proven to be more difficult. Initially, clinical indicators such as weight loss and progression of opportunistic infections were used to stage HIV disease, but many asymptomatic AIDS patients could not be identified (Murai H 1989. “American Journal of Medicine” 86: 533-8; Kaplan J. E. et al., 1992 “Journal of Acquired Immunity Dependency. Syndrome No. 5: p.565-70 (Murray, HW et al, 1989. Am. J. Med. 86: 533-8; Kaplan, JE et al, 1992. J. Acquir. Immune Defic. Syndr. 5 : 565-70). With the availability of effective diagnosis using prophylactic antibiotics and antiretroviral therapy, useful clinical markers of HIV disease status became necessary.

 Several candidates for serum markers were studied in the late 1980s and early 1990s, including the HIV p24 antigen (Lanji J. M. et al., 1987 “AIDS” No. 1: p. 155-9 (Lange, JM et al, 1987. AIDS 1: 155-9)), anti-HIV antibody, serum IgA (immunoglobulin A; Stewartlander B. et al., 1993 “AIDS” No. 7: p.813-22 (immunoglobulin A; Schwartlander, B. et al, 1993. AIDS 7: 813-22)), IgG, IgM, Β-2 microglobulin (Anderson Earl et al., 1990, “Archives. Of Internal Medicine "No. 150: p. 73-7 (Anderson, RE et al, 1990. Arch. Intern. Med. 150: 73-7)), neoptrin (Bogner J. R. et al., 1998). Year “Cliniche Vohensh (Klinische Wochenschrift) No. 66: p. 1015-8 (Bogner, JR et al, 1988. Klin. Wochenschr. 66: 1015-8)), adenosine deaminase and soluble interleukin 2 receptor (IL-2R) ) Level (Lanji JM. Et al., 1988, “Anal Medicine Intern (Paris)” 139: p.80-3; Fafey J. L. et al., 1990, “The New England・ Journal of Medicine ”322: pp. 166-72; Savin C. A. et al., 1994“ British Journal of Himatology ”No. 86 (2): pp. 366-71;・ J.M., et al., 1995, “Journal of Acquired Immun Deficiency Sindro And Human Retrovirology ”, No. 8: p. 266-72; Planetera T. et al., 1998“ Clinical Chemistry and Laboratory Medicine ”, No. 36: p. , JM et al, 1988. Ann. Med. Interne (Paris) 139: 80-3; Fahey, JL et al, 1990. N. Engl. J. Med. 322: 166-72; Sabin, CA et al, 1994 Br. J. Haematol. 86 (2): 366-71; Zabay, JM et al, 1995. Acquir. Immun. Defic. Syndr. Hum. Retrovirol. 8: 266-72; Planella, T. et al, 1998 Clin. Chem. Lab. Med. 36: 169-73)). All these markers were found to be useful in certain circumstances, but did not show a direct quantifiable correlation with progressive disease. The clinical utility of periodic measurements using these markers has not yet been proven, and there are technical limitations that make some assays difficult. With the exception of the p24 test, these markers were not widely adopted in actual clinical practice.

  Some laboratories temporarily introduced the p24 antigen test because the serum concentration of p24 was considerably higher in most patients who met the criteria determined clinically as AIDS than in asymptomatic patients. However, an anti-p24 antibody response occurs in many patients, after which p24 can circulate throughout the body as an antibody-bound immune complex. If these immune complexes are not acid or heat denatured prior to testing, p24 levels are underestimated in commercial assays, limiting the clinical use of p24 (Nadal Dee et al., 1999). “Journal of Infectious Diseases” No. 180: p. 1089-95 (Nadal, D. et al, 1999. J. Infect. Dis. 180: 1089-95)). Furthermore, even after dissociation of immune complexes, the reason for this is unknown, but p24 levels in many advanced AIDS patients are negligible (Ercoli El. Et al., 1995.11: p. 1203-7 (Ercoli, L , et al 1995.11: 1203-7)). Because of these problems with p24 measurement and the time-consuming assay, there is a direct correlation with disease—especially CD4 counts and HIV-RNA levels—more reliable tests have become desirable prognostic markers.

  Early studies on HIV-1 infection demonstrated that the most stable indicator of clinical disease was CD4-positive T cell count. Long-term studies have also shown a clear correlation between CD4 count <200 cells / μL and death, with a slow but stable decrease in CD4 count at a rate of up to 50 CD4 positive cells / μL per year. (Fahey J. L. et al., 1990, The New England Journal of Medicine No. 322: pp. 166-72 (Fahey, JL et al, 1990. N. Engl. J. Med. 322: 166-72)). Based on these research results, the Center for Disease Control and Prevention reclassifies the definition of AIDS, and includes patients whose CD4-positive T cell count has decreased to less than 200 cells in AIDS (the Center for Epidemiology and Prevention Center “Mobility and Motority”).・ Weekly Report (MMWR) ”1992; 41 (RR-17): p. 1-19 (Centers for Disease Control MMWR 1992; 41 (RR-17): 1-19)), CD4 positive T cell count Quickly became a totem in the HIV clinical laboratory. In an accompanying study, the absolute number of CD4, the percentage of total lymphocytes that were CD4 positive (“CD4 percent”), and the ratio of CD4: CD8 were all useful markers for disease progression (Movidity and Motor Center for Disease Control and Prevention. (Written by the Weekly Report of MMWR) 1997; 46: p. 1-29 (Centers for Disease Control and Prevention MMWR 1997; 46: 1-29)), especially in infants and children .

  In 1995 and 1996, a series of announcements by Meroles et al. Significantly changed the nature of HIV clinical testing in developing countries (Loveday C. and A. Hill 1995 “Lacent” 345: p. 790-1; Melols J. W., et al., 1995 “Anals of Internal Medicine” No. 122: p.573-9; Melols J. W., et al., 1997 “Science” 1996.272. : Pp. 1167-70 [Eratum Appears in Science 275: 14]; Melols J. W. Drew, et al., 1997, "Anals of Internal Medicine" 126: 946-54 (Loveday, C. and A. Hill. 1995. Lancet 345: 790-1; Mellors, JW et al, 1995. Ann. Intern. Med. 122: 573-9; Me llors, J. W. et al, 1997. Science 1996.272: 1167-70 [Erratum appears in Science 275: 14]; Mellors, J. W. et al, 1997. Ann. Intern. Med. 126: 946-54)). It was found that the amount of HIV-RNA present in the serum-"viral load"-was directly correlated with clinical disease. Ancillary studies have confirmed that serum HIV-RNA levels are the single most important indicator of the course of subsequent HIV infection in individual patients (Cooms Earl W., et al. 1996, Journal of Infectious Diseases "174: 704-12; O'Brien W. A. et al. 1997" Anals of Internal Medicine "126: p. 939-45; O'Brien W. A. 1996, The New England Journal of Medicine, No. 334: p. 426-31, Yerry S. et al, 1998, Archives of Internal Medicine, 158: p. 52 (Coombs, RW et al, 1996. J Infect. Dis. 174: 704-12; O'Brien WA et al, 1997. Ann. Lntern. Med. 126: 939-45; O'Brien, WA et al, 1996. N. Engl. J. Med. 334: 426-31; Yerly, S. et al. 1998. Arch. Intern Med. 158: 247-52)). Viral load measurement quickly became the standard for treatment and became a criterion for treatment with antiretroviral drugs. By 1996, official HIV treatment guidelines encouraged HIV-infected patients to measure CD4 counts and HIV-RNA levels every 3 months (Carpenter C. J. et al. 1996, Journal. Of American Medical Association ”276: p.146-54; Sarg MS, et al.“ Nature Medicine ”No. 2: p.625-629 (Carpenter, CCJ, et al. 1996. JAMA 276: 146-54; Saag, MS et al. 1996. Nat. Med. 2: 625-629)).

  The effects of liver enzymes, especially alanine aminotransferase and aspartate aminotransferase, among those liver enzymes, are increased in AIDS patients and people who are positive for HIV serum reaction. (Fan C. M. et al., 1988 “Clinical Chemistry”, Volume 34 (12): p. 2574-6 (Huang, CM et al, 1988. Clin. Chem. 34 (12): 2574) -6)). In addition, many of the drugs used for HIV treatment, and many of the infections associated with HIV, cause liver damage. Aminotransferases cause α-amino group removal from L amino acids and are mainly present in the liver as described above. These α-amino groups are removed during the oxidative degradation reaction and recycled for use in amino acid biosynthesis or excreted as urea. By transamination, all amino groups are collected from different amino acids into L-glutamic acid. Glutamic acid leads the amino group to either the biosynthetic or excretory pathways (ie the human urea cycle). These two enzymes are important not only for the diagnosis of HIV progression but also for the diagnosis of liver damage and are regularly monitored in most HIV-infected patients.

  The cost of testing outperforms the detection sensitivity, specificity and clinical utility of various HIV diagnostic tests. The cost for immunoassays against serum proteins such as HIV antibodies, p24 antigen, β-2 microglobulin typically ranges from $ 3 to $ 15 per test, with newer simple tests being 1 per test It can be as little as a dollar. However, immunoassay requires reading with a spectrophotometer, costs about $ 8,000 per photometer, and requires a stable power supply. Even if reliable prognostic markers that can be measured by immunoassay have been developed, these costs are an impediment to use in resource-poor countries.

  Standard treatment tests used to monitor HIV treatment in the United States and Europe are significantly different from those described above. CD4 counts are significantly more expensive than immunoassays and cost between $ 20 and $ 100 per test. CD counting requires a machine called a flow cytometer, typically $ 30,000- $ 100,000 with a laser and requires a lot of power and maintenance. Three types of HIV-RNA tests are currently approved for viral load measurement. All are based on amplification (eg, PCR, bDNA, NASBA) and cost more than $ 100 per test and require a highly research environment and highly skilled personnel. Nucleic acid amplification costs $ 80,000, a thermocycler, and requires a device that requires stable power and temperature control.

  In a resource-poor environment devastated by HIV, there is no room for financially, structurally, or technically to regularly measure CD4 count and HIV viral load using these existing technologies. Therefore, even if HIV medications are free and hospital, clinic, or community-based treatment programs are implemented, which patients should be treated and which treatments are being treated There are currently no widely applicable systems for determining how to be monitored. Last year, a conference attended by laboratory scientists, HIV clinicians, international health policy makers, and ministers of health in various countries was imminent about affordable HIV diagnosis and treatment monitoring tools in developing countries. Necessity was examined. Three methods emerged from these meetings: validation of new surrogate markers for HIV disease (eg, hemoglobin levels); improvements to the scarce environment of existing tests and assays; and CD4 counts, HIV-RNA measurements And the development of new technologies for other tests in light of standardized treatment in the developed world.

  While efforts to identify new surrogate markers—diagnostic criteria, lymphocyte counts, and hemoglobin levels—have limited success, they have only repeated the limitations of these methods in the United States and Europe in the late 1980s. Assays that measure the action of reverse transcriptase as a surrogate for viral load have been somewhat promising, but are still costly and technically problematic. As a more effective method, the p24 assay was modified to develop an inexpensive and rapid immunoassay for the heat-denatured p24 antigen (HDp24) (Ledergelvale B. et al., 2000 “Journal of Infectious. “Dizzies”, 181: 1280-8; Pascal A. et al., 2002, “Journal of Clinical Microbiology”, Volume 40: pp. 2472-5; World Health Organization AIDS Countermeasure Program, 1994 “AIDS” 8: WHO1-WH04 (Ledergerber, B., et al. 2000. A Infect. Dis. 181: 1280-8; Pascual, A., et al. 2002. J. Clin. Microbiol. 40: 2472- 5; World Health Organization Global Program on AIDS. 1994. AIDS 8: WH01-WH04)). In preliminary studies in a scarce environment, this assay is 85% sensitive, 100% specific, costs $ 8 and correlates with HIV-RNA levels (Pascal A. et al., 2002 “Journal of "Clinical Microbiology" No. 40: p. 2472-5 (Pascual, A. et al. 2002. J. Clin. Microbiol. 40: 2472-5)). The HDp24 assay is currently the best candidate for an immunoassay that can be readily introduced for therapeutic monitoring, but the technical complexity of cost and processing is a concern and further assay refinement and validation studies are needed.

  Efforts to develop affordable flow cytometers for low-cost CD4 counts have similarly limited success (World Health Organization AIDS Countermeasure Program, 1994 “AIDS” 8: WHO1-WH04; Lyamuya) • EF et al. 1996 “Journal of Immunological Method” No. 195: p. 103-12; Sherman GE et al., 1999 “Journal of Immunological Method” No. 222: p.209-17; Janosie G. et al., 2002 “Cytometry” No. 50: p.78-85 (World Health Organization Global Program on AIDS. 1994. AIDS 8: WHO1-WH04; Lyamuya, EF, et al. 1996. J. Immunol. Methods 195: 103-12; Sherman, GG et al. 1999. J Immunol. Methods. 222: 209-17; Janossy, G., et al. 2002. Cytometry 50: 78-85)). Another CD4 counting method using magnetic bead separation (Dynabeade (R), Dynal), Oslo, Norway) and manual cell counting is a small scale at a major reference research facility in West Africa. (Diaguboga S. et al., 2002, Oral Presentation No. WeOrB1342, 14th International AIDS Conference (Diagbouga, S. et al, 2002. Oral Presentation # WeOrB1342, XIVth International AIDS Conference)). Whether this method can be extended to national treatment programs at local and regional levels, and the final cost of the assay is still unclear. Currently, the Dynabeade method (US Pat. No. 4,910,148) appears to be the only method of CD4 counting that can be readily implemented at a reasonable cost, but the technical requirements of the assay (ie, the CD4 positive for the Dynabyde method) T cells need to be quantified with antibodies labeled with magnetic microspheres and separated with a magnetic separator), making their use difficult. Dinavidee CD4 counts are currently available, but the cost will probably range from $ 3 to $ 15 per assay, and future HIV testing in resource-poor environments due to technical limitations will be left to the new technology Will be.

  Other technologies have also been recognized and actively explored, and going may be available. General improvements in immunochromatographic techniques have also been reflected in HIV testing. A cartridge and dipstick test to measure HIV-1 antibody for HIV diagnosis in a one-step lateral flow format is available (Ketema F. et al., 2001 “Journal of Acquired Immun Defiance” Sea Syndrome No. 27: p. 63-70 (Ketema, F. et al, 2001. J. Acquir. Immune Defic. Syzdr. 27: 63-70)). Similar assays that measure not only HIV antibodies but also serum proteins that can serve as surrogate markers for clinical HIV disease are currently under evaluation. Nuclear amplification tests using single tubes are being actively developed for many applications, including HIV monitoring in resource-poor environments. These tests still require sophisticated techniques, but the cost of HIV-RNA measurement may be reduced to less than $ 10 (De Bar MP, et al., 2001 “Journal of Clinical Micro. Biology, 39: p. 1895-902; O Sea-Wye, "Monitoring and Diagnostic Tools for the Management of Antiretroviral Therapy in Resources-Poor. Settings, Bethesda, MD, November 11-13, 2001 (de Baar, MP et al, 2001. J. Clin. Microbiol. 39: 1895-902; Oh, CY. Monitoring and Diagnostic Tools for the Management of Antiretroviral Therapy in Resource-Poor Settings, Bethesda, Md., Noveber 11-13, 2001)).

  Commercially available equipment, Biometric Imag 2000 (R) and 4T8 cartridge (R) (Ogoman M. 1998 "Conference on the Laboratory Science of H.I. buoy" 97-111 (O ' Other automated methods using Gorman, M. 1998 Conference on the Laboratory Science of HIV 97-111)) also involve cumbersome technical requirements. For example, specimen scanning is performed by centrifugation, and detection requires laser scanning to create a peak emission profile that requires further processing.

  World attention has been devoted appropriately to the shortage of HIV drugs available, but there remains little touch on the affordable HIV diagnostic technology required. Given the availability of funding for antiretroviral therapy, there is an urgent need for HIV clinical tests designed for resource-poor environments. The new test takes into account not only severe cost constraints but also the lack of refrigeration, power and technical support in the scarce environment where the majority of 40 million people living with HIV worldwide live. There is a need.

  As mentioned above, the most stable indicators of the extent of damage to the immune system by HIV and the risk of clinical development of AIDS are the absolute number of CD4 positive T cells (percentage of total lymphocytes that are CD4 positive) and the CD4: CD8 ratio. (Mellors J. W. et al., 1995 (Mellors JW et al., 1995)). The significance of these markers for monitoring HIV-1 progression is supplemented by US guidelines, which start treatment in all patients with CD4 positive cells <350 cells / mm3 and are CD4 positive It is recommended that the count should be measured every 3 to 6 months during treatment.

  Over the last decade, efforts to develop an appropriate CD4 positive counting method in a scarce environment have not been successful. Part of the reason for not being successful is that traditional assays require sophisticated and expensive equipment, stable power, and advanced expertise. In particular, assays that monitor HIV infection based on available, price-sensitive, reliable, power-independent microchips are highly desirable.

Means to try to solve the problem

  A microchip-based screening method for HIV specimens will now be described. The method of the present invention uses an improved microchip flow cell and uses a “target analyte” associated with HIV such as cells (eg, CD4 cells), nucleic acids (eg, HIV-RNA), proteins (eg, liver enzymes), etc. Are captured directly from whole blood. The captured specimen is imaged using an advanced fluorescence detection system that combines a still image capturing system such as a CCD digital camera and stable fluorescence signal transmission. Accurate analyte quantification can be performed from the whole blood of a sample collected with a fingerstick without the need for additional processing or sample amplification.

  Current specimen screening methods often employ flow cytometry. Flow cytometry requires processing using a dynamic fluid flow including the target analyte. In the method of the present invention, the subject analyte is passed through a unique flow cell with one or more cavities that place and capture the capture agent, thereby providing another volume of whole blood and / or plasma. Separate the analyte from the phase. The captured analyte is then optically imaged using a light source, one or more dichroic filters, and a detector capable of imaging the immobilized analyte (ie, a “still image”). In this way, the method of the present invention provides efficient capture and still image capture of a small molecule analyte of interest.

  In one embodiment, CCD digital imaging is used to take a still image. Small molecule detection is facilitated by a combination of efficient capture, still image capture, and stable fluorescence signaling. Because small molecules can be easily captured and detected, blood sample volume, reagents, and power requirements can be significantly reduced. Flow cytometry, on the other hand, relies on capturing signals from the analyte moving through the flow stream, and thus requires more cumbersome processing methods and detection equipment.

  In one embodiment, the present invention provides a microchip-based HIV diagnostic method, in which assays for a variety of specimens (eg, CD4 positive T cells, HIV-RNA, p24 antigen, liver enzyme) are performed. It is implemented with a single microchip that is optically readable with a portable battery-powered microchip reader.

Accordingly, the present invention relates to a method for detecting an HIV-related analyte in a blood sample, the method comprising:
a) passing an HIV-related analyte bound to a fluorescent luminescent conjugate through a flow cell comprising one or more cavities, wherein the one or more cavities are provided with a filter, whereby the analyte is placed on the filter To separate the specimen from the blood,
b) illuminating the analyte with a wavelength suitable for excitation of the conjugate;
c) imaging conjugates emitting fluorescent signals digitally using a detector;
including.

  In one embodiment, the filter is a polycarbonate porous membrane.

The present invention relates to a method for determining the ratio of CD4 positive T cells to CD8 positive T cells in a sample blood, the method comprising:
a) passing CD4-positive and CD8-positive T lymphocytes bound to different fluorescent luminescent conjugates through a flow cell comprising one or more cavities, wherein the one or more cavities include Comprising a filter, whereby CD4 positive and CD8 positive T lymphocytes are immobilized on the filter,
b) illuminating lymphocytes with a wavelength suitable for excitation of each conjugate;
c) digitally imaging each conjugate that emitted a fluorescent signal using a detector, and d) determining the ratio of CD4 positive cells to CD8 positive cells.

The present invention relates to a method for detecting HIV-RNA in a blood sample, the method comprising:
a) passing a blood sample containing HIV-RNA through a flow cell comprising one or more cavities, wherein the one or more cavities contain a complementary nucleotide sequence bound to a fluorescent compound. Including a filter having
b) forming a binding complex between the HIV-RNA on the filter and the complementary nucleotide sequence, enhancing the signal emitted from the fluorescent compound by the formation of the binding complex;
c) illuminating the complex with a wavelength suitable for excitation of the fluorescent compound, and d) using a detector to digitally image the fluorescence signal emitted by the complex.

The present invention is a method for detecting an HIV-related analyte in a blood sample, the method comprising:
a) passing an HIV-related analyte bound to a fluorescent conjugate through a flow cell comprising one or more, wherein the flow cell comprising one or more has an appropriate binding affinity for the analyte Including agarose beads with biological factors, thereby immobilizing the specimen on the beads and separating the specimen from the blood;
b) illuminating the analyte at a wavelength suitable for excitation of the conjugate;
c) digitally imaging the conjugate that emitted the fluorescent signal using a detector.

The present invention relates to a method for determining the ratio of CD4 positive T cells to CD8 positive T cells in a blood sample, the method comprising:
a) passing CD4 positive and CD8 positive T lymphocytes, each conjugated with a different fluorescent luminescent conjugate, through a flow cell comprising one or more cavities, wherein the one or more cavities contain CD4 Agarose beads having binding affinity for either positive and CD8 positive T lymphocytes are included, thereby immobilizing the lymphocytes on the beads and separating the specimen from the blood.

The present invention relates to a method for detecting HIV-RNA in a sample blood, the method comprising:
a) Passing a blood sample containing HIV-RNA through a flow cell comprising one or more cavities, wherein the one or more cavities are complementary nucleotide sequences bound to a fluorescent compound. Comprising agarose beads having
b) forming a binding complex between the nucleotide sequence complementary to the HIV-RNA on the bead, wherein the signal emitted from the fluorescent compound is caused by the binding of the complementary nucleotide sequence to the HIV-RNA. Enhanced,
c) illuminating the complex with a wavelength suitable for excitation of the fluorescent compound, and d) using a detector to digitally image the fluorescent signal emitted by the complex.

  Other aspects of the invention are described in or are obvious from (and within the scope of the invention) the following disclosure.

  Here, systems and methods relating to the analysis of fluids containing one or more target analytes are described. As used herein, “analyte” refers to a biological factor that is efficiently immobilized, detected, and quantified using this system and includes nucleic acids, proteins, and cells. In one example, the specimen in the present invention is associated with HIV infection (ie, the quantification of the specimen indicates the presence or severity of HIV infection in the subject).

  The system may be used for either liquid or gaseous fluids. In some embodiments, the system may generate a pattern that indicates a diagnosis for both an individual specimen and a mixture of specimens. In some embodiments, the system is made up of a plurality of chemically reactive particles that form an ordered array that can rapidly detect many different types of analytes simultaneously. As an aspect of the system, the microfabrication process may be applied to form the array, which allows the system to be manufactured without cost.

In one example analyte detection system, the system, in some examples, includes a light source, a flow cell with a sensor array, and a detector. The sensor array, in some embodiments, is in the form of a support member configured to hold various capture agents, such as particles that react chemically in an ordered array (herein referred to as “particles”). Examples of particles include, but are not limited to, functionalized polymer beads, agarose beads, dextrose beads, polyacrylamide beads, porous glass beads, metal oxide particles (eg, silicon dioxide (SiO ₂ ) or aluminum oxide ( Al ₂ O ₃ )), polymer thin films, metal quantum particles (eg, Si, Ge, GaAs, etc.).

  A detector (eg, charge coupled device “CCD”, photodiode, CMOS imager, or other device that reacts to light) may be placed under the sensor array to enable data acquisition. In another example, a detector may be placed on the sensor array to acquire data from reflections of light emitted from particles or other capture agents.

  Light emitted from the light source passes through the sensor array and passes through the bottom side of the sensor array. The light modulated by the particles may pass through the sensor array and reach a proximally spaced detector. Evaluation of the optical change may be completed by visual inspection or using the detector itself or the detector in combination with an optical microscope. The microprocessor may be coupled to a detector or a microscope. The fluid supply system may be coupled to a support member of the sensor array. The fluid supply system is configured to direct the sample into and out of the sensor array in some embodiments.

  The particles, in some embodiments, have both the ability to bind to the analyte of interest and generate a modulated signal. These particles, in some embodiments, are elements that generate a signal that can be detected in the presence of an analyte. The particles may generate an optical (eg, absorbance or reflectance) signal or a fluorescence / luminescence signal upon irradiation of the specimen. The particles can be associated with a biological agent that has both the ability to bind the analyte of interest and generate a modulated signal. The bioagent can be bound to a label (eg, a complementary HIV sequence labeled with a fluorescent compound). The bioagent may have the ability to bind to the analyte of interest. When the biological agent is bound to the target analyte, a modulated signal may be generated when the label is exposed to light. Biological factors are naturally occurring or man-made and may be formed by logical design or combinatorial techniques. Examples include, but are not limited to, DNA, RNA, protein, enzyme, oligopeptide, antigen, antibody. Whether natural or artificial biological factors are used may be selected based on the ability to bind to the specimen in a specific manner.

  In one example, the bioagent is coupled to a luminescent compound such as a fluorophore. In another example, the analyte itself can be bound to the luminescent compound. These conjugated entities are referred to herein as “fluorescence detection conjugates”.

  In one embodiment, the sensor array system includes a number of particles, where a bioagent (eg, receptor, antibody, DNA, RNA sequence) associated with one or more polymer beads or agarose beads. You may have. In some embodiments, the bioagent is selected for its ability to interact with the analyte. This interaction may take the form of binding / association with, for example, an analyte. The support member may be made of any material that can pass the appropriate wavelength of light while supporting the particles.

  In one example, the bioagent and label may be coupled with a polymeric resin. Upon capture, structural changes occur in the presence of the analyte, thereby causing changes in the local microenvironment of the label. This change may cause a change in the spectroscopic properties of the label. Depending on the signaling protocol used, different signals may be generated upon interaction with the sign. Such protocols may include absorbance, fluorescence resonance energy transfer, and / or fluorescence quenching.

  In one embodiment, the sensor array system includes a number of particles contained within individual cavities. The particles may have biological factors bound to polymer or agarose beads. A suitable biological factor can be selected by interaction with the target analyte. This interaction may take the form of binding / association with the analyte. The support member may be made of any material that can pass the appropriate wavelength of light while supporting the particles. The support member may include a plurality of cavities. The cavity may be formed so that at least one particle is substantially contained within the cavity.

  The support member may have a plurality of cavities, which are also referred to herein as “microwells”. The cavity can be formed by a micromachining technique known in the prior art. The cavity may be formed, for example, in a pyramidal hole so that at least one capture agent is accommodated in the cavity. In one embodiment, the capture agent is a particle having agarose beads or filters, each capable of binding to a biological agent.

  The sensor array may include a cover layer. The cover layer may be located slightly above the sensor array, thereby forming a path between the sensor array surface and the cover layer. The cover layer may be spaced a little apart so that the cover layer prevents particles from falling out of the cavity in the sensor array and allows fluid to enter the cavity through a path formed between the sensor array and the cover layer. Allow inflow.

  In some embodiments, the cavity may be configured to allow fluid to pass through the cavity during use, while the cavity is configured to retain a capture agent within the cavity as the fluid passes through the cavity. . For example, the cavity may contain agarose beads bound to a biological agent (eg, HIV gp41 and / or gp120 antigen, HIV p24 antigen, and hepatitis B surface antigen) having a specific binding affinity for the subject HIV specimen. Good.

  A suction machine may be attached to the cavity. A suction machine may be applied to the entire sensor array. Alternatively, a suction device may be attached to the cavity and the cavity may be in a vacuum state. The suction device may be any device that can provide a pressure differential and move fluid. The suction device may apply a suction force to any fluid in the cavity. Examples of the suction device include a previously sealed vacuum chamber, a vacuum pump, a vacuum line, and a suction type pump.

  In an embodiment, the optical detector may be integrated into the bottom of the support member rather than utilizing a separate detection device. A microprocessor may be attached to the optical detector so that fluid evaluation can be performed without the use of a separate detection component. Furthermore, a fluid supply system may also be incorporated into the support member. By integrating the detector and fluid supply system with the support member, a compact and portable analyte sensing system can be formed.

  Sensitive sensor arrays (eg, CCD or CMOS) may be used to measure changes in optical properties that occur upon bio / chemical agent binding. This array may be coupled to a filter, light source, fluid supply, and micromachined particle container, thereby creating a functional sensor array. In one embodiment, data acquisition and processing may be performed using existing CCD or CMOS technology. A CCD or CMOS detector may be configured to measure white light, ultraviolet light, and fluorescence. As other detectors, a photomultiplier tube, a charge induction device, a photodiode, a photodiode array, a microchannel member, or the like may be used.

  In one embodiment, naturally occurring or man-made biological factors are bound to macromolecules or agarose beads to create particles. Particles, in some embodiments, have both the ability to bind to the analyte of interest and to generate a detectable signal. In some embodiments, the particles generate an optical signal when bound to the analyte of interest.

  A variety of natural and man-made biological factors include, but are not limited to, polynucleotides (eg, aptamers), peptides (eg, enzymes and antibodies), and receptors. A polynucleotide is a relatively small piece of DNA that may be obtained by sequential construction of DNA sequences. Peptides may include natural peptides such as antibodies or enzymes, or may be synthesized from amino acids.

  In one example, the particles are based on natural biopolymers but may have non-natural biopolymers that are chemical structures composed of non-natural linking units. For example, polythiourea and polyguanidine have structures similar to peptides but may be synthesized from diamines (ie, compounds containing at least two amine functional groups) rather than amino acids.

  In one embodiment, the bioagent may be combined with a polymeric resin. At the time of capture, if there is an analyte, a chemical reaction occurs and a signal is generated. The label may be bound to a bioagent or polymer bead. A chemical reaction may cause changes in the local microenvironment of the label to change the spectroscopic properties of the label. This signal may be generated using various signaling protocols. Such protocols may include absorbance, fluorescence resonance energy transfer, and / or fluorescence quenching. Examples of combinations include, but are not limited to, peptide-protease, polynucleotide-nuclease, oligosaccharide-oligosaccharide cleaving agent.

  Further details regarding these systems are set forth in the following US patent applications, all of which are hereby incorporated by reference: US patent application Ser. No. 09 / 287,248, entitled “Fluid Based Analysis of the Invention”.・ Fluid Based Analysis of Multiple Analyzes by a Sensor Array ”; US patent application Ser. No. 09 / 354,882, entitled“ Sensor Arrays for the Measurement and. “Identification of Multiple Analyzes in Solutions”; US patent application Ser. No. 09 / 616,355, entitled “Detection System Based.” On An Analyte Reactive Particles (Detection System Based on an Analyte Reactive Particles); US patent application Ser. No. 09 / 616,482, title of the invention “General Signaling Protocols for Chemical Receptors in Immobilized MATERIALS” for Chemical Receptors in Immobilized Matrices); US patent application Ser. No. 09 / 616,731, entitled “Method and Appalatis for the Delivery of Samples to a Chemical Sensor Array” U.S. patent application Ser. No. 09 / 775,342, entitled “Magnetic-based Placement and Retention of Sensors Elements in a Sensor”. Array (Magnetic-Based Placement and R "etention of Sensor Elements in a Sensor Array"; US patent application Ser. No. 09 / 775,340, entitled “Method and System for Collecting and Transmitting Chemical Information”. Collecting and Transmitting Chemical Information); US patent application Ser. No. 09 / 775,344, entitled “System and Method for the Analysis of Bodily Fluids” U.S. Patent Application No. 09 / 775,353, Title of Method "Method of Preparing a Sensor Array"; U.S. Patent Application No. 09 / 775,048, Title of Invention "System for Transferring Fluid Samples Through • System for Transferring Fluid Samples Through A Sensor Array ”(US Patent Application Publication No. 2002-0045272-A1); US Patent Application No. 09 / 775,343, entitled“ Portable Sensor Array System ” Array System); and US patent application Ser. No. 10 / 072,800, title of invention “Method and Apparatus for the Confinement of Material in a Micromachined Chemical Sensor Array” (Method and Apparatus for the Confinement of Materials in a Micromachined Chemical Sensor Array) ".

  In another embodiment, the capture agent comprises a membrane based flow sensor configured to accommodate a filter in which a cavity in the fluidics device is disposed within the fluidics device. Samples, particularly cells such as lymphocytes and other leukocytes, are larger in size than the filtration pores, and are thus captured by the flow cell and immobilized on the membrane. The captured analyte may be directly analyzed or treated with a visualization compound prior to imaging.

  As described herein, various analytes may be captured and analyzed using membrane-based flow sensors. Samples of particular interest for detection include lymphocytes, monocytes, and other immune system cells.

  FIG. 1 is an enlarged view of a membrane-based flow sensor 100. The flow sensor 100 includes a membrane 110 sandwiched between at least two members 140 and 150. Members 140 and 150 are configured to allow fluid to enter and pass through membrane 110. The members 140 and 150 are also configured to capture the analyte with the membrane 110 before detecting the analyte. A variety of different materials may be used for the membrane 110 such as, but not limited to, Nuclepore (product name: Nuclepore®) track etch membrane, nitrocellulose, nylon, cellulose acetate and the like. In general, the material used for the membrane 110 should be resistant to non-specific binding of antibody and staining solution used during the visualization and detection process. Further, the membrane 110 is made of a material that is inert to various reagents, buffers, and solvents. The membrane 110 may have a plurality of submicron pores that are precisely and uniformly arranged. By using a membrane having uniformly arranged holes, it is possible to control more reliably than fluid flow or specimen separation.

  The members 140 and 150 are made of a material that is substantially transparent to the wavelength of light used for analyte detection. For example, when it is necessary to use ultraviolet rays in the specimen detection method, the member 140 is preferably made of a material that is substantially transparent to ultraviolet rays. The member 140 may be made of any material suitable for meeting the detection method criteria. Transparent materials that may be used to form member 140 include, but are not limited to, polymers such as glass, quartz glass, and acrylate polymers (eg, polymethyl methacrylate). In some embodiments, the upper member 140 and the lower member 150 are both made of a transparent material. By using a transparent material for the upper member and the lower member, detection can be performed via a membrane-based flow sensor.

  As shown in FIG. 1, the film 110 is sandwiched between the upper member 140 and the lower member 150. Lower member 150 and / or upper member 140 may have a recess configured to hold the membrane. For example, in FIG. 1, the lower member 150 includes a recess 152 configured to receive the membrane 110 with other accompanying components used to support or encapsulate the membrane 110. The recess or cavity may be etched into the upper member 140 and / or the lower member 150 using standard etching techniques.

  Referring to FIG. 1, the lower member 150 includes a first recess 152 configured to receive the member support 130. The lower member also includes a second recess 154. The second recess is configured such that the member support 130 does not enter the second recess. The second recess may have a ridge portion disposed in the vicinity of the member support 130 so that the member support 130 is placed on the ridge portion. Alternatively, as shown in FIG. 1, the size of the second recess may be smaller than the size of the member support 130. In either case, at the time of assembly, the member support 130 is prevented from entering the second recess 154, thereby providing a cavity under the member support 130. The second recess 154 may be used to collect fluid passing through the member support 130 prior to draining from the system.

  The member support 130 is configured to support the membrane 110 during use. The member support 130 may be formed from a porous material so that fluid can pass through the member support. The size of the holes in the member support 130 should be sized so that the fluid can pass through the member support 130 at a rate that is greater than or equal to the rate at which the fluid can pass through the membrane 110. In one embodiment, the hole in member support 130 is larger than the hole in membrane 110, but the hole should not be too large. One function of the member support 130 is a function of supporting the film 110. Therefore, it is desirable that the hole of the member support 130 is sufficiently small so that the membrane 110 does not bend during use. The member support 130 may be formed from various materials such as, but not limited to, polymeric materials, metals, and glass. In one embodiment, a polymeric material (eg, celcon acrylic) may be used as the material for the member support 130. In addition, the member support 130 helps to keep the membrane planar during use. By maintaining the film in a plane, the specimen can be captured and detected on a single focal plane, and the specimen detection can be simplified.

  As described above, when the membrane-based flow sensor 100 is assembled, the membrane 110 may be placed on the member support 130. In some embodiments, the gasket 120 may be placed on top of the membrane 110. A gasket may be used as a fluid leak proof seal between the member 130 and member 140 and the membrane 110. The gasket may prevent fluid leakage from the system during use.

  The upper member 140 may include a fluid inlet portion 160. A fluid to be analyzed is introduced into the apparatus 100 via the fluid inlet 160. The fluid inlet 160 may pass through a portion of the upper member 140. In some embodiments, the path 162 may be formed in the upper member 140 and the tube 164 may be inserted into the path 162. The path 162 may be returned near the center of the upper member to supply fluid to the upper surface of the membrane 110.

  The lower member 150 may be provided with a fluid outlet 170. The fluid introduced into the device 100 via the fluid inlet 160 passes through the upper member 140 and passes through the membrane 110. The fluid is then collected in the recess 154. The fluid outlet 170 may be penetrated through a part of the lower member 150. In some embodiments, the path 172 may be formed in the lower member 150 and the tube 174 inserted into the path 172. Path 172 may be arranged to receive fluid collected in cavity 154 during use.

  Depending on the situation, a cleaning fluid outlet 180 may be formed in the upper member 140. The cleaning fluid outlet 180 is configured to receive fluid that passes or traverses the membrane 110 during a cleaning operation. The cleaning fluid outlet 180 may penetrate a part of the upper member 140. In some embodiments, the path 182 may be formed in the upper member 140 and the tube 184 may be inserted into the path 182. Path 182 may be arranged to receive the fluid used to clean membrane 110 during use.

  The membrane 110 is selected from materials that can filter the analyte of interest from the fluid flow. For example, assuming that lymphocytes are the target specimen, it is desirable that the filter can remove lymphocytes from the fluid flow. A suitable membrane may include a plurality of pores that are significantly smaller than the size of the analyte of interest.

  The membrane may be formed from a variety of materials known in the prior art. In one embodiment, the membrane 110 may be a track-etched Nuclepore (trade name: Nuclepore ™) polycarbonate porous membrane. Nukulipore membrane is commercially available from Whatman plc. The thickness of membrane 110 is about 5-10 microns. The membrane 110 has a plurality of holes. The pore size ranges from about 0.2 μm to about 12 μm. The porous membrane filter can be supported by a plastic screen disk (trade name: Celcon®).

If the target analyte is white blood cells (eg, CD4 lymphocytes), a disposable membrane filter is desirable for capture. For example, a polycarbonate track etching film having 3 μm pores is fixed in the cavity of the flow cell, and a lymphocyte capturing means having a surface area of about 80 mm ² and an internal volume of about 20 μL is prepared. Under such conditions, deformed red blood cells (which have a similar diameter as lymphocytes, but typically more than 1000 times in quantity) easily pass through the pores under appropriate fluid flow, while leukocytes are trapped. And deposited on a single imaging focal plane. Separation of red blood cells reduces autofluorescence and allows imaging of white blood cells taken directly from whole blood without additional sample processing.

  FIG. 2 shows an embodiment of a membrane-based flow sensor disposed in the housing 200. The upper member 140, the gasket 120, the membrane 110, the member support 130, and the lower member 150 may be installed in the housing 200. The housing 200 may include a membrane based fluid sensor. A cap 210 may be used to hold the membrane based fluid sensor in the housing 200. The cap 210 may be provided with a window so that the film 110 can be observed. Positioning the fluid inlet portion 160, the fluid outlet portion 170, and the cleaning fluid outlet 180 within the housing 200 allows the membrane-based fluid sensor 100 to be easily accessed by extending from the housing 200.

  FIG. 3 shows an overall schematic view of a membrane-based analysis system. The analysis system includes a plurality of pumps (p1, p2, p3, p4). The pump is configured to supply sample (p1), visualization reagents (p2 and p3), and membrane cleaning fluid (p4) to the membrane-based fluid sensor 100 during use. After passing the reagents, cleaning fluid, and visualization agent through the pre-filters (f1, f2, f3, f4), the fluid is sent to the membrane-based fluid sensor 100. The prefilter is used to eliminate large particulate matter that can clog the membrane 110. The nature and size of each prefilter may be optimized to efficiently capture particulate matter that accumulates while preventing large dust particles or clogging. The pre-filter f1 is configured so that the sample reaches the membrane-based fluid sensor 100 after filtering the sample. The prefilter f1 is configured to pass the target specimen while preventing the inflow of some particles unrelated to the target specimen. For example, spores having a size smaller than the pores of the prefilter f1 pass through the prefilter and are captured by the membrane-based fluid sensor 100. The fluid passes through the manifold after passing through the prefilters f1-f4. In some embodiments, the membrane-based fluid sensor 100 includes a single inflow line. At the manifold, different fluid lines are connected to the inflow line of this single membrane-based fluid sensor 100.

  The fluid is introduced into the fluid inlet of the membrane-based fluid sensor 100 after passing through the manifold. When appropriate, the detector 250 is used to determine whether the membrane based fluid sensor 100 is capturing an analyte. As shown in FIG. 5, a detector may be placed over a portion of the membrane-based fluid sensor 100 so that the detector captures an image of the membrane. For example, the detector may be installed so that an image of the membrane can be taken through a window in the membrane-based fluid sensor 100. Detector 250 may be used to acquire an image of particulate matter captured on membrane 110. Acquiring an image may include generating a “digital map” of the image. In some embodiments, detector 250 may include a sensitive sensor array (eg, CCD or CMOS). This sensor array may be connected to a filter, a light source, and a fluid supply unit to form a functional sensor array. In one embodiment, data acquisition and processing may be performed using existing CCD or CMOS technology. In some embodiments, light is decomposed into three color components, red, green, and blue. Evaluation of optical changes may be accomplished by visual inspection (eg, with a microscope) or using a microprocessor (“CPU”). For fluorescence measurements, a filter may be placed between detector 250 and membrane 110 to remove the excitation wavelength. As shown in FIG. 3, a microprocessor may be used to control the pumps and valves. A microprocessor may also be used to control, for example, LEDs or other light sources, CMOS or CCD imaging components and imaging processing software.

The analyte detection system may be operated in different modes based on opening and closing of the valve. The system configuration for the “inflow” mode is shown in FIG. In this mode, fluid is moved from the manifold to the membrane-based fluid sensor 100 to allow analyte capture or developer addition. Analysis fluid may be introduced into the membrane-based fluid sensor 100 via the fluid inlet 160. During operation in the “inflow” mode, valve v ₁ is in the closed position and restricts fluid flow through the cleaning fluid outlet 180. As such, fluid may be forced through the membrane based fluid sensor 100 and out of the sensor via the fluid outlet 170. Valve v ₂ is in the open position, fluid is allowed to flow in the object container. Valve v ₃ is in a closed position, thereby suppressing the flow of fluid to the cleaning fluid supply line.

The specimen detection system may be operated in a “lateral membrane cleaning” mode, as shown in FIG. In this mode, fluid is flowed over the membrane collection surface to clean the membrane. This allows the membrane to be reused for the next inspection. A membrane cleaning fluid may be introduced into the sensor 100 via the fluid inlet 160. During the “lateral membrane cleaning” operation, the outlet valves v ₂ and v ₃ are in the closed position to inhibit fluid flow through the fluid outlet 170. Also, closing the outlet valves v ₂ and v ₃ suppresses the flow of fluid through the membrane of the sensor 100. Therefore, the fluid flowing into the sensor 100 is forced out of the sensor 100 via the cleaning fluid outlet 180. The valve v2 is in the open position so that fluid flows into the drainage reservoir via the cleaning fluid outlet 180. Since the fluid is restrained from flowing through the membrane, analytes and other particles collected on the membrane are washed from the membrane, making the membrane more usable.

  The sample detection system may be operated in a “backwash” mode, as shown in FIG. During the backwash operation, fluid outlet 170 is used to introduce fluid into the analyte detection system, while wash fluid outlet 180 is used to allow fluid out of the apparatus. The “reverse” flow of fluid through the cell cleans the membrane. In some embodiments, the valve may be configured with a cleaning fluid introduced via a fluid outlet 170 as shown in FIG. Specifically, the valves v1 and v3 are opened and the valve v3 is closed.

  Either lateral membrane cleaning or back-flow treatment may be used to remove analyte and other particles from the membrane. Both methods of cleaning the membrane surface may be enhanced using ultrasound or mechanical agitation. In use, the analyte in the fluid sample is trapped in the membrane because it is larger than the hole in the membrane. After use, the analyte tends to be randomly distributed throughout the membrane. The specimen located between the holes on the membrane is more difficult to remove than the specimen located on or near the pores of the membrane because the force of the backwash fluid may not strike the specimen. During backwashing and lateral washing operations, ultrasound, which is mechanical agitation, may be used to enhance removal of trapped analytes. Both cleaning methods move the specimen over the membrane surface, increasing the chance that the specimen will hit the cleaning fluid column passing through one of the holes.

  An analyte detection system may be used to determine the presence of an analyte in the fluid system. An example of an analyte determination process for a fluid sample is shown in the flowchart of FIG.

  Prior to analyzing the sample, a background sample may be collected and analyzed. Solid specimens are usually collected and stored in a liquid fluid. The liquid fluid used for sample preparation may be analyzed to determine whether an analyte is present in the fluid. In one embodiment, a sample of liquid fluid used to collect a solid specimen is introduced into the specimen detector to determine background “noise” generated by the fluid. During background collection, the particles collected on the membrane are observed to determine the level of particulate matter in the liquid fluid. In some embodiments, the particles collected by the membrane during the collection stage may be treated with a visualization agent to determine whether an analyte is present in the liquid fluid. Information collected in background tests may be used when analyzing collected samples to reduce false positives.

  As described herein, after the background sample is collected, the membrane may be cleaned using either backwashing or lateral cleaning. After cleaning the membrane, the system may be used to analyze samples for solid analytes.

  As the collected sample passes through the porous membrane, the porous membrane traps any analyte having a size larger than the size of its pores. Particle collection may continue for a predetermined time or until all of the collected sample has passed through the membrane.

  After collection, the specimen collected on the membrane may be analyzed with a detector. In some embodiments, the detector may be a camera that captures an image of the membrane. For example, the detector may be a CCD or CMOS camera. Analysis of the particles captured by the membrane may be performed by analyzing the size and / or shape of the particles. The presence of a given analyte may be pointed out by comparing the size and / or shape of the particles captured on the membrane with the known particle size and / or shape. The specimen also reacts with various visualization agents (eg, colored dyes and fluorescent dyes). Analyze the analyte captured on the membrane with an appropriate detector. The presence of particles having the relevant color and / or fluorescence may indicate the presence of the analyte for testing purposes.

  In one example, it is desirable to detect CD3, CD4, CD8 cells in whole blood. Prior to applying to the flow cell, the whole blood sample can be incubated with anti-CD3 and anti-CD4 antibodies conjugated with a fluorophore for an appropriate period of time (eg, 8 minutes). The detection power can be increased by culturing in advance. The supply of the test sample to the flow cell can be adjusted using a fluidics controller (eg, a peristaltic pump).

  A digital image from one region of the lymphocyte capture membrane is conjugated with two different absorption filters, eg, an antibody filter conjugated with AleXa488 (identifies CD4 positive T lymphocytes and emits a green signal) and AleXa647 Acquired using a separate filter for identified antibodies (identifies CD3-positive T lymphocytes and emits a red signal). The two images are then automatically synthesized in a digital format to allow the subject CD3 positive / CD4 positive T lymphocytes (ie, CD4 cells displayed in yellow) to become CD4 positive / CD3 negative monocytes (green). Display) and CD3 positive / CD4 negative T lymphocytes (displayed in red).

  Analyzing the particles may indicate the presence of the target analyte in the sample. In this case, the particles may be washed from the membrane and delivered out of the system for further inspection. Additional tests may utilize techniques such as culture or ELISA techniques that can more accurately determine the presence of a particular specimen. Alternatively, the particles may be sent to a sensor array for further inspection as described herein. If there is no effective amount of analyte on the membrane, the membrane may be washed and other samples analyzed.

  User-defined threshold criteria may be set to indicate the probability that one or more specific analytes are present on the membrane. This criterion may be set based on one or more of the various features of the image. In some embodiments, this reference may be set based on a pixel or color fingerprint (fingerprint) preset for a particular specimen. Features that can be used include, but are not limited to, the size, shape, and color of the portion of the material on the image, the aggregate that the material creates, the fluorescence intensity of the material, and the like.

  The membrane system may comprise a computer system (not shown). The computer system may include one or more software applications that can process the digital map generated using the detector. For example, an inspection image may be compared to a predefined optical fingerprint using a software application available on a computer system. Alternatively, software available on the computer system may be used to determine if the count exceeds a predefined threshold limit.

  Detectors may be used to acquire images of analytes and other particulate matter captured on the membrane. You may analyze the image acquired with the detector based on the preset reference | standard. A positive result may indicate the presence of bacteria. The inspection criteria may be based on various features of the image, such as, but not limited to, the size, shape, aspect ratio, color, etc. of a portion or portion of the image. By applying the test standard, the target specimen may be distinguished from the background particulate matter. During the analysis, the sample may flow continuously from the fluid supply system.

  In some embodiments, it may be deduced from positive results that the fluid contains a particular analyte. If the image shows a positive result for the test criteria, the fluid sample may be subjected to a verification test or a specific test. On the other hand, if the image shows a negative result with respect to the test criteria, the membrane may be rinsed and the method described above may be performed on fluid from another sample.

  During the specimen test, the sample may be introduced into the specimen detection apparatus. The trigger parameter may be measured to determine when to introduce the visualization agent into the specimen detection device. The trigger parameter measurement may be performed continuously or may be initiated by the user. Alternatively, the staining solution may be introduced into the specimen detection apparatus immediately after the sample is introduced.

  In one embodiment, the trigger parameter may be the time that has elapsed since the introduction of fluid at the controlled flow rate into the analyte detection device. For example, the staining solution may be introduced 20 seconds after the fluid sample is introduced into the specimen detection apparatus at a flow rate of 1 ml per minute. In another embodiment, the trigger parameter may be a pressure drop across the membrane. The pressure drop across the membrane may be determined using pressure transducers on both sides of the membrane.

  In another example, the trigger parameter may be autofluorescence of an analyte captured by the membrane. The detector may be switched on until the signal from the analyte autofluorescence reaches a predefined level. In yet another embodiment, filtering software may be used to create an autofluorescence data map of the material on the membrane, excluding pixels that contain blue or red spectral band colors. A data map may be used to calculate values for autofluorescent particles in only the “pure green” portion of the visible spectrum.

  In some embodiments, a positive result may be estimated if the trigger parameter exceeds a certain value without adding staining solution. For example, a positive result may be estimated if the autofluorescence value exceeds twice the value suggesting that a staining solution has been added. In such cases, no staining solution may be added and a confirmation test may be performed on the sample.

  If the value of the trigger parameter is lower than the value that suggests that the process proceeds directly to the confirmation test, but exceeds the value set for performing the addition of the staining solution, the staining solution may be introduced into the specimen detection apparatus.

  Fluid sample collection may include sample collection from a solid, liquid, or gas. In some embodiments, the sample may be extracted from air taken from the subject environment in aerosol form, and the aerosol then converted to a hydrosol. For example, particles may be collected from a 500 L air sample and precipitated into about 0.5 mL of liquid. US Pat. No. 6,217,636, inventor McFarland, the title of the invention “TRANSPIRATED WALL AEROSOL COLLECTION SYSTEM AND METHOD” Although incorporated herein by reference as if fully set forth herein, it describes a system that uses pore walls to collect particulate matter from a gas stream in a liquid.

  The following examples describe and explain the invention in more detail, but do not limit the invention.

Example Example 1
Microchip-based assay for measurement and quantification of CD4 positive T cells Design and development of flow cell analysis chamber Chip consisting of individually assignable microwells on a single silicon or plastic microchip Based sensor arrays have been developed. Microwells were made and optimized by plastic molding methods, and the resulting structure was anisotropically etched to function as a small reaction vessel and analysis chamber. Each microwell has a volume of about 30 nanoliters (nL); sufficient samples to perform multiple assays with 1 microliter (μL) of fluid. The microwell has a pyramidal depression shape and has an opening that allows fluid to flow through the analysis chamber and is placed at the bottom of the microwell or within the microwell. Enables optical analysis of reactions occurring in microbeads or membrane filters. Current chips contain up to 100 microwells on one very small chip (FIGS. 7 and 8).

  The microchip is fixed in a microfluidic flow cell. A modified version of the flow cell is encased in a three-piece stainless steel casing with a flat platform and is attached to a permanently circular vertical support, which is replaced by a replaceable (screw-type) cap. They are connected (FIG. 9). Inside the metal casing, there are upper and lower inserts made of circular polymethyl methacrylate (PMMA). These inserts allow fluid to pass through an integrated stainless steel tube (Microgroup Inc., Medway, Mass.) And into the fluid flow cell via the fluid supply system (upper left inlet) ) And drain (upper right and lower exit) (FIG. 9). In addition to providing a drain tube, a PMMA lower insertion section includes a lymphocyte capture membrane (for example, a 3.0 m microporous filter (product name: Nuclepore®), Whatman plc., Clifton. (New Jersey), a plastic screen disk that functions as a support, and a gasket between the membrane and top insert prevents leakage and ensures that all samples are fed into the flow cell and filtered through the membrane. The top outlet is used in communication with the lateral fluid flow to remove air bubbles as needed.

Fluid supply system
Early studies used a peristaltic pump to supply both sample and wash buffer to the flow cell. Subsequent designs used partially automated fluid delivery systems. The version uses a platform with two OEM-made plastic pumps, each pump (v ₁ and v ₂ respectively) using 0.031 "silicone tubing and a flow rate of 0.046 per minute. Connected to a pinch valve that can supply the flow cell at ~ 0.92 mL.Integrated software uses appropriate combination of pump and valve to direct supply and wash cycle of colored blood sample.Filtered with capture membrane The sample and wash fluid contain red blood cells and are collected in the drainage reservoir A third valve (v ₃ ) is placed after the top outlet of the flow cell and is used with p ₂ to remove bubbles formed during the assay. Remove.

  Subsequent studies used a 3.0 micron capture membrane to optimize the flow cell microfluidics to retain lymphocytes and remove red blood cells. As shown in FIG. 10, erythrocytes were effectively filtered without losing lymphocytes retained in these membranes.

Optical station and image capture and analysis
The analytical flow cell was placed on the stage of an Olympus Corporation BX2 microscope equipped with a 4X objective and a high pressure mercury lamp as the light source. Focus was maintained on a constant surface on the membrane during the entire assay. Visualization of (red) lymphocytes stained with AleXa647 was performed using a Cy5 ^TM filter cube (620 nm excitation, 660 longpass beam splitter dichroic mirror, 700 nm fluorescence, Olympus), while stained with AleXa488 ( Green) Lymphocytes were visualized using a fluorothiocyanate (FITC) filter cube (excitation at 480 nm, 505 longpass beam splitter dichroic mirror, 535 ± 25 nm fluorescence, Olympus). A large number of images were taken in different areas of the membrane with an electric stage.

For each study object, a total of five non-overlapping regions of the lymphocyte capture membrane of the flow cell were mounted on a microscope 12-bit digital video camera (DVC) 1312C (DVC Company, Austin, TX) charge binding Images were taken with a device (CCD). Each region was imaged twice, using the Cy5 ^TM filter cube for fluorescence detection by AleXa488 once and the FITC filter cube for fluorescence detection by AleXa647 once. Except for dose-response experiments, these two images were combined to create a single digital image prior to analysis.

  Images were analyzed using a custom algorithm developed with a commercially available image processing software package (Image-Pro Plus, Media Cybernetics, Inc.). In this algorithm, thresholds were set for red, green and blue intensities, and lymphocytes were optimally defined for background fluorescence; lymphocytes were also characterized by their outline (size and shape). Background correction was performed to remove autofluorescence, and objects identified as presumed positive cells were enhanced by standard image processing protocols to blur sharp edges. The cells thus identified were then counted in an automated manner and the results were calculated using spreadsheet software (Excel, Microsoft Corporation, Redmond, WA) with CD4 + CD3-, CD4 + CD3 +, CD8 + CD3-, CD8 + CD3 + For each antibody combination used, such as CD4 + CD8 + cells, the number was recorded.

CD4 positive lymphocyte detection
As described above, a microchip system with a 3.0 μm porous filter was adapted for optimal lymphocyte capture to set a standard for microchip CD4 positive counts. These studies were performed using peripheral blood mononuclear cells (PBMC) collected from healthy subjects. For these experiments, buffy coat (lymphocyte fraction in whole blood) was collected and PBMCs were separated by Ficoll density gradient centrifugation. T lymphocyte subsets were> 98% pure from PBMC by an immunomagnetic separator (Miltenyi Biotec Inc., Auburn, Calif.) Erythrocyte Rosette Formation (Stem Cell Technologies Inc.) Collected at The subset was resuspended in RPMI solution at a stock concentration of 106 cells / mL and used within 24 hours after production. The cells were stored frozen in RPMI solution of 10% dimethyl sulfoxide (DMSO) and thawed immediately before use. In all cases, cells were counted and assessed for viability immediately prior to use.

  Purified CD4 cells were labeled with AleXa488 conjugated anti-CD4 antibody (Molecular Probes Inc., Eugene, Oreg.) For 5 minutes at room temperature and then controlled at 0.3 mL per minute. At a rapid rate, various dilutions (range 0-200,000 CD4 positive cells / mL) were introduced into the flow chamber. Samples were injected into the flow chamber by hand or automatically using high performance / pressure liquid chromatography (Pharmacia) driven by Unicorn version 3.0 software (Amersham).

  After sample introduction, the chamber was washed with 2-5 mL of phosphate buffered saline (PBS) and then introduced at 1.2 mol per minute. This capacity is 10,000 dead volume, which removes unnecessary blood components such as platelets and red blood cells and greatly reduces the background signal. The chamber was then imaged by fluorescence microscopy. FIG. 10A shows an original image obtained by increasing the number of CD4 cells from 0 to 200,000 cells / mL (which corresponds to the physiological range seen in AIDS patients with advanced symptoms). FIG. 10B shows a linear correlation (R2 = 0.999) between the number of cells in the sample and the light intensity as measured from the digital image by pixel analysis. This proved that the number of lymphocytes labeled with a fluorescent marker can be accurately measured and detected using a microchamber and a digital image analysis system.

Example 2
Measurement of CD4: CD8 ratio and CD4 positive ratio from whole blood Whole blood assay for detecting CD4 positive and CD8 positive cells and determining CD4: CD8 ratio in a short time based on the microchip described in Example 1 (No erythrocyte lysis, buffer addition, or sample addition) was developed. Flow parameters and sample dilution were adjusted to show an optimal flow rate of 0.8 mL per minute and a 1:20 whole blood dilution.

  Blood was collected from healthy subjects or HIV-infected subjects by venipuncture at Massachusetts General Hospital and Botswana Harvard Institute in Habarone, Botswana. For measurement of the CD4: CD8 ratio and CD4 positive ratio, 33 microliters of whole blood was mixed with 3 microliters of antibodies against CD3, CD4, CD8 conjugated with a staining fluorophore. An optimal staining protocol was set for each antibody used in this study by observing in vitro reactions and results on slides. The antibody was applied to a microcentrifuge tube before use (room temperature, 3,000 × g for 2 minutes) and the supernatant reaction was used for staining. This process ensured removal of fluorescent particulate matter that could be captured by the membrane and thus interfere with imaging. Stained blood samples were suspended to 500 μL with PBS and introduced directly into the flow cell followed by washing with 2 mL PBS. Thereafter, images of labeled cells captured on the membrane were acquired and analyzed as described above. In a subset of samples, cells that were not retained on the filter were collected in a drain reservoir and counted to determine capture efficiency. For some experiments, after acquiring images, fixative (2% paraformaldehyde / 2.5% glutaraldehyde) was introduced into the flow cell and then rinsed with PBS. The filter was removed from the flow cell and fixed with osmium oxide (OSO 4) vapor for 90 seconds, then dehydrated with ethanol (EtOH) / Hexasamethyldisilazane (HMDS) and subjected to scanning electron microscopy (SEM).

  The total time from blood collection to image acquisition, analysis and results was less than 15 minutes. Early work on fluorophores suggested that light stability and pH were important determinants of the fluorescence signal in this system. Therefore, various fluorophore antibody combinations were evaluated in a pilot study, but for all subsequent studies, only AleXa class (type) fluorophores were used. Since only antibodies conjugated to AleXa488 and AleXa647 against the CD antigen could be used, this system was limited to two-color imaging. FIG. 11 shows a series of representative original images collected from HIV infected subjects using a whole blood assay. In FIG. 11A, an antibody conjugated with AleXa488 labels CD4 positive cells in green in subject B38, an HIV-infected subject with a CD4 count of 961 cells / mL as determined by flow cytometry. To focus on the same area of the microchip using a second emission filter, all T lymphocytes are colored red with an antibody against CD3 conjugated with AleXa647 (FIG. 11B). By automatically synthesizing the image, the system converts the subject CD3-positive CD4-positive T lymphocytes, displayed in yellow, into CD4-positive CD3-negative monocytes (green) and CD3-positive CD4-negative T lymphocytes (red). ) (FIG. 11C). By masking unnecessary cells automatically, as shown in FIG. 11D, only CD4-positive CD3-positive cells to be counted can be left.

  Using a custom algorithm developed within the image processing software environment of the system, the total number of lymphocytes of each subtype was automatically calculated from the original image and sent to a data file.

  In this algorithm, thresholds for red, green and blue intensities were set for optimal definition of lymphocytes for background fluorescence; characterization by lymphocyte profile (size and shape) was also performed. Background correction was performed to remove autofluorescence, and objects identified as presumed positive cells were enhanced by standard image processing protocols to blur sharp edges. The cells thus identified were then counted in an automated manner and the results were calculated using spreadsheet software (Excel, Microsoft Corporation, Redmond, WA) with CD4 + CD3-, CD4 + CD3 +, CD8 + CD3-, CD8 + CD3 + For each antibody combination used, such as CD4 + CD8 + cells, the number was recorded.

  For each study subject, five non-overlapping images were acquired and used to count the number of cells, increase the sample cell population, and improve accuracy.

Correlation with flow cytometry As shown in FIG. 12, the ratio of CD4 cells to the total number of T lymphocytes obtained from the microchip system (the number of CD4-positive CD3-positive cells / the total number of CD3-positive cells) and the CD4: CD8 ratio (CD4 The results (percentage of cells / percent of CD8 cells) were compared for each study subject with the results obtained in parallel by flow cytometry.

  The two methods were in great agreement with the absolute CD4 cell range, the ratio of CD4 cells to the total T lymphocyte count, and the CD4 / CD8 ratio all seen with HIV infection. As for CD4 absolute numbers, the method was compared by the Passing-Bablok23 method, and there was an excellent correlation with flow cytometry (FIG. 12A), and the Bland-Altman plot 24 had zero bias and a good coincidence limit of 95% ( FIG. 12B) is shown respectively. The calculated Pearson correlation was r = 0.92. Similar results were obtained from both the CD4 count (FIGS. 12C-D) and the CD4 / CD8 ratio (FIGS. 12E-F) reported as a percentage of total CD3 positive T lymphocytes in CD4 cells. The coincidence limits were more closely related to the assessment of CD4 cell percentage and CD4: CD8 ratio, but with respect to absolute CD4 numbers. This probably reflects the expected sample supply capacity variation in the prototype, affecting the absolute count but not the ratio. In addition, because of the significant delay in processing of some samples with respect to flow cytometry rather than microchip assays, some microchip results are actually closer to the “true” value than flow cytometry results. There may be. There is almost agreement between the two methods in the correlation between the subset of 15 subjects and the absolute number of CD4 below 200 cells / mL.

Example 3
Microbead immunoassay Agarose microbeads were coated with various pathogen specific antigens such as HIV gp41 / gp120, HIV p24, hepatitis B virus surface antigen. Beads coated with an antibody against a specific antigen were placed in a well of a silicon microchip, and then a serum sample with a known antibody titer was run through the microchip system and detected with a fluorescence microscope using a Cy2-labeled secondary antibody. In FIG. 13, one such experimental result is shown; sera with antibodies against HIV and hepatitis B that are easily detected using the microchip method.

  FIG. 15 shows the results of another microbead experiment in which HIV p24 antigen was detected in human serum containing 100 pg / mL HIV p24 antigen. The same principle can be applied to all immunoassays, such as liver enzymes.

Example 4
One method of demonstrating the effectiveness of antiretroviral drugs to suppress the clinical development of HIV-RNA measurement and quantification AIDS from whole blood of samples collected with a fingerstick is to monitor the viral load before and after treatment. It is done. Viral load measurement often helps doctors determine when to start antiretroviral therapy. For example, if the CD4 count is on the borderline (350 cells / mm ³ ), a high viral load will be the basis for initiating treatment with antiretroviral therapy, and a low viral load will be the basis for watching. In addition, viral load measurement is a guideline for doctors to design appropriate treatment courses.

  Currently, HIV viral load is generally quantified by detection using one of several real-time fluorescence detection methods after a PCR-based method with viral sequence amplification using a thermocycler. The However, this assay requires a skilled technician with stable power and additional processing. These requirements greatly limit their use in resource-poor environments. In such a scarce environment, the need for affordable HIV clinical testing is enormous. The assay presented here describes the results of an assay for HIV-RNA that includes a simple, inexpensive, power-independent microchip.

Quantitative parameters For these assays, a microchip-based sensor assay; consisting of individually assignable agarose beads coupled to a biotinylated molecular indicator DNA capture probe was made. The probe was selectively placed in a micromachined cavity localized on a silicon microchip wafer. Samples containing target DNA and RNA pass through these micro-chambers, bind to the capture sequence and emit a fluorescent signal. To verify the concept of a microchip-based HIV-RNA essay, an HIV-specific molecular label (clade B-type gag, tgcagaatgggatagattg) was attached to the microbeads and a sample containing complementary HIV-RNA sequences was obtained from femtomole. The microchip was introduced in a concentration range up to nanomolar. HIV-RNA is annealed in the sample with the capture sequence attached to the microchip, spreading the molecular beacon, resulting in bright fluorescence. As shown in FIG. 15A, the reference signal (without HIV-RNA) does not lead to an increase in the distance of the molecular beacon, and therefore shows only the minimum fluorescence signal. In contrast, as shown in FIG. 15B, adding a sample containing 100 pM HIV-RNA results in a bright fluorescent image.

  To set the specificity of the HIV-RNA assay, the beads are coated with 18 base-paired DNA oligomers, each different by one nucleotide, and labeled with a fluorescent dye that clearly identifies the capture sequence for each probe of interest. The experiment was carried out by mixing in a 1: 1: 1 ratio in the solution. The chip was then inspected against this equimolar solution and binding was detected with a fluorescence microscope. As shown in FIG. 16, sequences labeled with fluorescent dyes bind only to their complementary target probes, and do not bind to target probes that are mismatched due to a single nucleotide difference.

  For the microchip-based assay described above, optimal binding of target RNA to molecular beacons and detection thresholds were set. Unlike other methods, this assay does not require 1) direct isolation of DNA from AIDS patients, followed by 2) sequence amplification using a thermocycler (eg, in vitro transcription for analytical RNA generation) .

Example 5
Microchip-based assay for liver enzyme measurement and quantification from whole blood samples collected with finger sticks. Two liver enzymes, alanine transamine (ALT) and aspartamide transamine (AST) are mild A moderate assessment is associated with liver failure, which is still the most common cause of death among AIDS-infected individuals. Liver damage is a major limiting factor in current HIV treatment efficacy, and monitoring of ALT and AST in AIDS patients is therefore regarded as important.

  Current serological analysis methods used for ALT and AST monitoring are not suitable for use in resource-poor environments because they are costly and require stable power and highly trained personnel. With the results presented here, the above constraints can be solved by describing a microchip-based essay that measures ALT and AST levels that is inexpensive and does not depend on power.

Quantitative Parameters For liver enzyme assays, antibodies specific for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) can be obtained and bound to microbeads. Samples containing known amounts of ALT and AST can be introduced into the chip to evaluate the sensitivity and detection threshold of the microchip assay. After introducing the sample, the chamber is cleaned and imaged under a fluorescence microscope. For fluorescence detection, a second sandwich antibody labeled with a fluorophore is introduced. The overall fluorescence intensity can then be correlated with standard ALT and AST concentrations to evaluate basic performance characteristics (linear range, detection threshold, reproducibility, effectiveness).

  Although detailed embodiments of the present invention have been described above, the present invention defined by the appended claims is not limited to the specific descriptions given above, but the spirit and scope of the present invention. It will be understood that as many obvious variations as possible are possible without departing from the invention.

DETAILED DESCRIPTION The following detailed description is given by way of example and is not intended to limit the invention to the specific embodiments described, which may be understood in conjunction with the accompanying drawings, wherein: To be incorporated here.
FIG. 3 is an enlarged view of a membrane-based flow sensor. Fig. 3 shows an embodiment of a membrane based flow sensor disposed within a housing. It is the schematic of the sample detection system in inflow mode. 1 is a schematic diagram of an analyte detection system in a lateral flow mode. It is the schematic of the sample detection system in backflow mode. It is a flowchart of a sample collection method. Shown is a 100 microwell microchip slightly smaller than a postage stamp. 1 is a schematic cross-sectional view of a microchip platform. The sample is injected at one end of the microchip and spreads uniformly through the reaction well. The reaction well has a volume of 30 nL. The sample can be washed to within 10,000 dead volumes using only 2 mL of wash buffer. At least two types of microchips can be used, one of which contains a polycarbonate membrane at the bottom of the microwell for collecting lymphocytes (ie for CD4 cell counting), and the other is each microwell (HIV-RNA or Agarose microbeads derivatized for enzyme measurement). 1 shows a pilot design of a flow cell and optical imaging system. The sample is introduced into the flow cell by a computer-controlled infusion minipump (FIG. 9A). Cells or specimens are retained in the microwells (FIG. 9B), while non-retained samples pass through the microwells and flow into the drainage reservoir (FIG. 9C). A light source (eg, a pressure mercury lamp) collects light at an appropriate focal plane at an appropriate wavelength or wavelength band onto an analyte captured in a microwell of the flow cell using a dichroic mirror. When the excitation light is captured by the analyte and its fluorescence emission detection conjugate and captured by the CCD digital camera, fluorescence emission due to the reaction occurs. The image can then be saved for analysis (FIGS. 9D, E). The dose response of CD4 lymphocytes is shown. Purified CD4 cells were labeled with anti-CD4 antibody conjugated with AleXa488, introduced into the flow cell in amounts ranging from 0 to 200,000 cells and imaged (FIG. 10A). There is a linear correlation between the concentration of CD4 cells in the sample and the intensity emitted by the labeled lymphocytes captured by these membrane filters (R ₂ = 0.999) (FIG. 10B). Raw data image and image processing for CD4 count. Digital images were obtained from single diluted whole blood specimens from HIV infected subjects with flow cytometry having an absolute CD4 count of 961 cells / μL. CD4 positive cells (T lymphocytes and monocytes) are stained green with an anti-CD4 antibody conjugated with AleXa488 (FIG. 11A). CD3-positive T lymphocytes are stained red with an anti-CD3 antibody conjugated with AleXa647 (FIG. 11B). By synthesizing these two images by digital processing, CD3 positive CD4 positive T lymphocytes (ie, “CD4 cells”) are displayed in yellow / orange, CD4 positive CD3 monocytes (green) and CD3 positive CD4 negative T lymphocytes. (Red) (FIG. 11D). A lymphocyte selection algorithm was applied to the synthesized images based on lymphocyte profiles defined by size, shape, homogeneity and CD4 positive and CD3 positive staining. Objects that do not match the CD4 positive CD3 positive lymphocyte profile are deleted, and the remaining objects are selected and digitally enhanced and counted (FIG. 11E). A protocol similar to CD8 cell count is utilized for each subject (FIG. 11F). Figure 2 shows a correlation study between the microchip-based system of the present invention and flow cytometry. Flow cytometry and the new microchip method are very consistent across the range of absolute CD4 numbers in HIV infection, the proportion of CD4 in the total number of T lymphocytes, and the CD4 / CD8 ratio. As for CD4 absolute numbers, the method comparison performed according to the Passing-Bablok method showed excellent correlation with flow cytometry (FIG. 12A), and the Bland-Altman plot 24 showed zero bias (bias 0) and good 95% agreement. Limits were shown (Figure 12B). The calculation result of the Pearson correlation coefficient is r = 0.92. Similar results were obtained for both the number of CD4 reported as a percentage of total CD3 positive T lymphocytes (FIGS. 12C-D) and the CD4: CD8 ratio (FIGS. 12E-F). The result of a microbead experiment is shown. Human serum with anti-HBsAg and anti-gp41 / 120 antibodies (at 1: 200) was cultured on antigen-coated microbeads and detected using fluorescent goat anti-human IgG antibody (at 1: 200). Results of HIV p24 antigen microbead assay are shown. Beads coated with antibodies against HIV p24 were placed in 4 microwells of a silicon microchip and reacted with human serum containing 100 pg / mL of HIV p24 antigen. Thereafter, Cy2-labeled anti-p24 secondary antibody was added and detected with a fluorescence microscope. The HIV-RNA assay is shown. HIV specific molecular beacons (clade B gag) were attached to the microbeads and samples containing complementary RNA (cRNA) sequences were introduced into the microchip. HIV-RNA sequences are annealed in samples with capture sequences attached to the microchip, spreading the molecular beacons and consequently brightly fluorescent. FIG. 15A shows a reference fluorescence signal. FIG. 15B shows the fluorescence signal after addition of a sample containing 100 pM complementary HIV-RNA sequence. The result of DNA base pair discrimination experiment is shown. The beads are coated with 18 base pair DNA oligomers, each different by one nucleotide. The capture sequence for each probe of interest is labeled with a clearly distinguishable fluorescent dye and mixed in a 1: 1: 1 ratio in solution. The chip is then tested against this equimolar solution and bound detected with a fluorescence microscope. Sequences labeled with fluorescent dyes bind only to their capture target probes and do not bind to target probes that are mismatched by a single nucleotide difference.

Explanation of symbols

110 Membrane 120 Gasket 130 Member Support 140 Upper Member 150 Lower Member 152 Recess 154 Second Recess 160 Fluid Inlet 162 Path 164, 174 Pipe 170 Fluid Outlet 172, 182 Path 180 Cleaning Fluid Outlet 200 Housing 210 Cap 250 Detection vessels _{v 1,} v 2, _{v 3} valve

Claims (38)

A method for detecting an HIV-related analyte in a blood sample, comprising:
a) passing an HIV-related analyte bound to a fluorescent conjugate through a flow cell comprising one or more cavities, wherein the one or more cavities are provided with a filter, whereby the analyte is Immobilizing on the filter to separate the specimen from the blood;
b) illuminating the analyte with a wavelength suitable for excitation of the conjugate;
c) imaging the conjugate emitting a fluorescent signal digitally using a detector;
A method characterized by comprising:   The method of claim 1, wherein the filter is a polycarbonate porous membrane.   The method of claim 1, wherein the detector is a CCD digital camera.   The method of claim 1, wherein the detector is a CMOS detector.   The method according to claim 1, wherein the specimen is a lymphocyte.   The method according to claim 1, wherein the lymphocyte is a CD4-positive T cell.   2. The method according to claim 1, wherein the lymphocyte is a CD8-positive T cell.   The method according to claim 1, wherein the lymphocyte is a CD3-positive T cell.   The method according to claim 1, wherein the specimen is a liver enzyme.   The method according to claim 9, wherein the liver enzyme is alanine aminotransferase.   The method according to claim 9, wherein the liver enzyme is aspartate aminotransferase.   2. The method of claim 1, wherein the fluorescent luminescent conjugate comprises an anti-CD4 antibody.   13. The method of claim 12, wherein the anti-CD4 antibody is an anti-CD4 antibody conjugated with Alexa488.   The method of claim 1, wherein the fluorescent luminescent conjugate comprises an anti-CD8 antibody.   2. The method of claim 1, wherein the fluorescent luminescent conjugate comprises an anti-CD3 antibody.   16. The method of claim 15, wherein the fluorescent luminescent conjugate is an anti-CD3 antibody conjugated with Alexa647.   The method according to claim 1, wherein the blood is collected from a blood sample collected with a finger stick. A method for determining the ratio of CD4 positive T cells to CD8 positive T cells in a blood sample, comprising:
a) passing CD4 positive and CD8 positive T lymphocytes, each bound to a different fluorescent luminescent conjugate, through a flow cell comprising one or more cavities, passing through one or more said cavities; Comprises a filter, whereby the CD4 positive and CD8 positive T lymphocytes are immobilized on the filter,
b) illuminating the lymphocytes with a wavelength suitable for excitation of each conjugate;
c) digitally imaging each conjugate emitting a fluorescent signal using a detector; and d) determining the ratio of CD4 positive cells to CD8 positive cells.
A method characterized by comprising: A method for detecting HIV-RNA in a blood sample, comprising:
a) passing a blood sample containing HIV-RNA through a flow cell comprising one or more cavities, wherein the one or more cavities contain a complementary nucleotide sequence bound to a fluorescent compound. Including a filter having
b) forming a binding complex between the HIV-RNA on the filter and the complementary nucleotide sequence, enhancing the signal emitted from the fluorescent compound by the formation of the binding complex;
c) illuminating the complex with a wavelength suitable for excitation of the fluorescent compound; and d) imaging the fluorescence signal emitted by the complex using a detector by digital processing.
A method characterized by comprising: A method for detecting an HIV-related analyte in a blood sample, comprising:
a) passing an HIV-related analyte bound to a fluorescent luminescent conjugate through a flow cell comprising one or more cavities, wherein the one or more cavities have a binding affinity for the analyte Including agarose beads with appropriate biological factors, thereby immobilizing the specimen on the beads and separating the specimen from the blood;
b) illuminating the analyte with a wavelength suitable for excitation of the conjugate;
c) digitally imaging the conjugate that emitted the fluorescent signal using a detector;
A method characterized by comprising:   21. The method of claim 20, wherein the detector is a CCD digital camera.   21. The method of claim 20, wherein the detector is a CMOS detector.   21. The method of claim 20, wherein the specimen is a lymphocyte.   21. The method of claim 20, wherein the lymphocyte is a CD4 positive T cell.   21. The method of claim 20, wherein the lymphocyte is a CD8 positive T cell.   21. The method of claim 20, wherein the lymphocyte is a CD3-positive T cell.   21. The method of claim 20, wherein the specimen is a liver enzyme.   28. The method of claim 27, wherein the liver enzyme is alanine aminotransferase.   28. The method of claim 27, wherein the liver enzyme is aspartate aminotransferase.   21. The method of claim 20, wherein the fluorescent luminescent conjugate comprises an anti-CD4 antibody.   32. The method of claim 30, wherein the anti-CD4 antibody is an anti-CD4 antibody conjugated with Alexa488.   21. The method of claim 20, wherein the fluorescent luminescent conjugate comprises an anti-CD8 antibody.   21. The method of claim 20, wherein the fluorescent luminescent conjugate comprises an anti-CD3 antibody.   34. The method of claim 33, wherein the fluorescent luminescent conjugate is an anti-CD3 antibody conjugated with Alexa647.   21. The method of claim 20, wherein the blood is collected from a blood sample collected with a finger stick. A method for determining the ratio of CD4 positive T cells to CD8 positive T cells in a blood sample, comprising:
a) passing CD4-positive and CD8-positive T lymphocytes, each conjugated with a different fluorescent luminescent conjugate, through a flow cell comprising one or more cavities, wherein one or more of the cavities has CD4 Comprising agarose beads with biological factors having binding affinity for either positive or CD8 positive T lymphocytes, thereby immobilizing the lymphocytes on the beads and separating the specimen from the blood;
b) illuminating the lymphocytes with a wavelength suitable for excitation of each conjugate;
c) digitally imaging each conjugate that emits a fluorescent signal using a detector, and d) determining the ratio of CD4 positive cells to CD8 positive cells,
A method characterized by comprising:   37. The method of claim 36, wherein the biological factor is an antibody. A method for detecting HIV-RNA in a blood sample, comprising:
a) passing a blood sample containing HIV-RNA through a flow cell comprising one or more cavities, wherein the one or more cavities contain a complementary nucleotide sequence bound to a fluorescent compound. Comprising agarose beads having
b) forming a binding complex between the HIV-RNA and the complementary nucleotide sequence on the bead, wherein the complementary nucleotide sequence binds to the HIV-RNA, whereby the fluorescence emission The signal emitted from the compound is enhanced,
c) illuminating the complex with a wavelength suitable for excitation of the fluorescent compound; and d) imaging the fluorescence signal emitted by the complex using a detector by digital processing.
A method characterized by comprising: